The hyperthermophilic nature of the metallo-oxidase from Aquifex aeolicus.
The stability of the Aquifex aeolicus multicopper oxidase (McoA) was studied by spectroscopy, calorimetry and chromatography to understand its thermophilic nature. The enzyme is hyperthermostable as deconvolution of the differential scanning calorimetry trace shows that thermal unfolding is characterized by temperature values at the mid-point of 105, 110 and 114 degrees C. Chemical denaturation revealed however a very low stability at room temperature (2.8 kcal/mol) because copper bleaching/depletion occur before the unfolding of the tertiary structure and McoA is highly prone to aggregate. Indeed, unfolding kinetics measured with the stopped-flow technique quantified the stabilizing effect of copper on McoA (1.5 kcal/mol) and revealed quite an uncommon observation further confirmed by light scattering and gel filtration chromatography: McoA aggregates in the presence of guanidinium hydrochloride, i.e., under unfolding conditions. The aggregation process results from the accumulation of a quasi-native state of McoA that binds to ANS and is the main determinant of the stability curve of McoA. Kinetic partitioning between aggregation and unfolding leads to a very low heat capacity change and determines a flat dependence of stability on temperature.
Fernandes
André T
AT
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, 2781-901 Oeiras, Portugal.
Martins
Lígia O
LO
Melo
Eduardo P
EP
D001419:Bacteria / Q000201:enzymology*; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D003300:Copper / Q000737:chemistry / Q000378:metabolism; D004795:Enzyme Stability / Q000187:drug effects; D019791:Guanidine / Q000737:chemistry / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D012542:Scattering, Radiation; D013816:Thermodynamics
D003300:Copper; D010088:Oxidoreductases; D019791:Guanidine